RNase H hydrolyzes RNA in RNA-DNA hybrids. RNase H activity appears to be ubiquitous in eukaryotes and bacteria. Although RNases H constitute a family of proteins of varying molecular weight, the nucleolytic activity and substrate requirements appear to be similar for the various isotypes. For example, all RNases H studied to date function as endonucleases exhibiting limited sequence specificity and requiring divalent cations (e.g., Mg2+, Mn2+) to produce cleavage products with 5′-phosphate and 3′-hydroxyl termini.
Recently, two human RNase H genes have been cloned and expressed. RNase H1 is a 286 amino acid protein and is expressed ubiquitously in human cells and tissues. The amino acid sequence of human RNase H1 displays strong homology with RNase H1 from yeast, chicken, E. coli and mouse. Human RNase H2 shares strong amino acid sequence homology with RNase H2 from C. elegans, yeast and E. coli. Although the biological roles for the human enzymes are not fully understood, RNase H2 appears to be involved in de novo DNA replication and RNase H1 has been shown in mice to be important for mitochondrial DNA replication.
The structure of human RNase H1 was shown to consist of a 73 amino acid region homologous with the RNA-binding domain of yeast RNase H1 at the amino-terminus of the protein and separated from the catalytic domain by a 62 amino acid spacer region. The catalytic domain is highly conserved with the amino acid sequences of other RNase H1 proteins and contains the key catalytic and substrate binding residues required for activity. Site-directed mutagenesis of human RNase H1 revealed that the spacer region was required for RNase H activity. Although the RNA-binding domain was shown not to be required for RNase H activity, this region was responsible for the enhanced binding affinity of the human enzyme for the heteroduplex substrate as well as the strong positional preference for cleavage exhibited by the enzyme. The RNA-binding domain of human RNase H1 is conserved in other eukaryotic RNases H1 and the highly conserved lysines at positions 59 and 60 of human RNase H1 have been shown to be important for binding to the heteroduplex substrate. The conserved tryptophan at position 43 was responsible for properly positioning the enzyme on the substrate for catalysis.
Human RNase H1 exhibits a strong positional preference for cleavage, i.e., human RNase H1 cleaves the heteroduplex substrate between 7 to 12 nucleotides from the 5′-RNA/3′-DNA terminus. Based on site-directed mutagenesis of both human RNase H1 and the heteroduplex substrate, the RNA-binding domain was shown to be responsible for the observed positional preference for cleavage. The RNA-binding domain of human RNase H1 appeared to bind to the 3′-DNA/5′-RNA pole of the heteroduplex substrate with the catalytic site of the enzyme positioned slightly less than one helical turn from the RNA-binding domain. Substitution of either the terminal 3′-DNA with a single ribonucleotide or 5′-RNA with a 2′-methoxyethoxy deoxyribonucleotide was shown to cause a concomitant 3′-shift of the first 5′-cleavage site on the RNA, suggesting that altering duplex geometry interferes with proper positioning of the enzyme on the heteroduplex for cleavage. Although the interaction between the RNA-binding domain and the heteroduplex substrate has been characterized, the mechanism by which the catalytic domain of RNase H1 recognizes the substrate has not been fully elucidated.
Human RNase H1 is a nuclease that cleaves RNA exclusively in an RNA/DNA duplex via a double-strand DNase cleavage mechanism. Neither double-strand RNA (dsRNA) or DNA (dsDNA) duplexes support RNase H1 activity. The observed structural differences between the RNA/DNA heteroduplex and dsRNA and dsDNA duplexes suggest a possible role for the helical geometry and the sugar conformation of the DNA and RNA in the selective cleavage of the heteroduplex substrate by human RNase H1. Specifically, the deoxyribonucleotides within dsDNA form a southern C2′-endo sugar conformation resulting in a B-form helical conformation, whereas ribonucleotides within dsRNA form a northern C3′-endo pucker and an A-form helical geometry. In contrast, the deoxyribonucleotides of the RNA/DNA heteroduplex have been shown to adopt an eastern O4′-endo sugar pucker resulting in a helical conformation where the RNA strand adopts A-form geometry and the DNA strand shares both the A- and B-form helical conformations. The conformational diversity observed for the DNA strand is likely a function of the intrinsic flexibility of the deoxyribonucleotide compared to RNA, and may also be important for human RNase H1 activity. DNA also differs from RNA in that the furanose ring of deoxynucleotide is much more flexible, i.e., exhibit a nearly symmetrical potential energy barrier for both south and north sugar conformations.
Consistent with these observations, heteroduplexes containing 2′-ara-fluoro deoxyribonucleotides, which have been shown to exhibit a sugar conformation comparable to DNA when hybridized to RNA, have also been shown to support RNase H1 activity. On the other hand, heteroduplexes consisting of RNA/2′-alkoxy modified deoxyribonucleotides, exhibiting C3′-endo sugar pucker and an A-form helical geometry when hybridized to RNA do not support human RNase H1 activity. It has previously been shown that both E. coli and human RNases H1 bind A-form duplexes (e.g., RNA/RNA, 2′-methoxyethoxy/RNA and 2′-methoxy/RNA) with comparable affinity to the DNA/RNA heteroduplex substrate but do not cleave the A-form duplexes. In this case, the size and position of the 2′-substituents of RNA and 2′-alkoxy nucleotides suggest possible steric interference with RNase H1 as the 2′-substituents are positioned within the minor groove of the heteroduplex; a region predicted to be the binding site for the enzyme. Alternatively, the sugar conformation and flexibility map play a decisive role in RNase H1 activity.
It can be seen that optimizing the cleavage of RNase H targets would be of great benefit. This invention is directed to this, as well as other, important ends.